U.S. patent application number 13/499758 was filed with the patent office on 2012-08-02 for human domain antibodies against components of the human insulin-like growth factor (igf) system.
Invention is credited to Weizao Chen, Dimiter S. Dimitrov.
Application Number | 20120195897 13/499758 |
Document ID | / |
Family ID | 43857387 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120195897 |
Kind Code |
A1 |
Dimitrov; Dimiter S. ; et
al. |
August 2, 2012 |
HUMAN DOMAIN ANTIBODIES AGAINST COMPONENTS OF THE HUMAN
INSULIN-LIKE GROWTH FACTOR (IGF) SYSTEM
Abstract
The invention provides antibodies or antibody fragments that
bind to insulin-like growth factor (IGF) 1 receptor (IGF-1R) or
IGF-2, as well as method of using the antibodies for inhibiting the
IGF-mediated signaling pathway, inhibiting IGF-1R signaling, and
treating cancer. The invention also provides a method of detecting
the presence of IGF-1R or IGF-2 in a sample using the inventive
antibodies and antibody fragments.
Inventors: |
Dimitrov; Dimiter S.;
(Frederick, MD) ; Chen; Weizao; (Frederick,
MD) |
Family ID: |
43857387 |
Appl. No.: |
13/499758 |
Filed: |
October 7, 2010 |
PCT Filed: |
October 7, 2010 |
PCT NO: |
PCT/US2010/051784 |
371 Date: |
April 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61249476 |
Oct 7, 2009 |
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Current U.S.
Class: |
424/134.1 ;
424/133.1; 424/178.1; 435/252.3; 435/252.31; 435/252.33;
435/252.34; 435/254.21; 435/320.1; 435/328; 435/69.6; 435/7.1;
436/501; 514/44R; 530/387.3; 530/391.1; 530/391.3; 530/391.7;
536/23.4; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/569 20130101; C07K 2319/00 20130101; G01N 2333/71
20130101; C07K 16/22 20130101; C07K 2317/565 20130101; C07K 16/2863
20130101; G01N 33/74 20130101; A61P 35/00 20180101; C07K 2317/73
20130101; C07K 2317/76 20130101; C07K 2317/92 20130101; C07K
2317/21 20130101; C07K 2317/33 20130101; A61K 39/39591
20130101 |
Class at
Publication: |
424/134.1 ;
530/387.3; 530/391.1; 530/391.3; 530/391.7; 536/23.53; 536/23.4;
424/133.1; 435/252.33; 435/252.31; 435/252.3; 435/252.34;
435/254.21; 435/328; 424/178.1; 514/44.R; 435/69.6; 435/320.1;
436/501; 435/7.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28; C07K 16/46 20060101
C07K016/46; C07K 19/00 20060101 C07K019/00; C12N 15/13 20060101
C12N015/13; C12N 15/62 20060101 C12N015/62; C12N 1/21 20060101
C12N001/21; C12N 1/19 20060101 C12N001/19; C12N 5/10 20060101
C12N005/10; A61K 31/7088 20060101 A61K031/7088; A61P 35/00 20060101
A61P035/00; C12P 21/02 20060101 C12P021/02; C12N 15/63 20060101
C12N015/63; G01N 33/566 20060101 G01N033/566; C07K 16/22 20060101
C07K016/22 |
Claims
1. A single domain antibody comprising: (a) SEQ ID NO: 1, SEQ ID
NO: 2, and SEQ ID NO: 3; (b) SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID
NO: 7; (c) SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) SEQ
ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; (e) SEQ ID NO: 17, SEQ
ID NO: 18, and SEQ ID NO: 19; (f) SEQ ID NO: 21, SEQ ID NO: 22, and
SEQ ID NO: 23; or (g) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO:
27.
2. The single domain antibody of claim 1 comprising SEQ ID NO: 4,
8, 12, 16, 20, 24, or 28.
3. A fusion protein or conjugate comprising the single domain
antibody of claim 1 and a cytotoxic agent, cell targeting motif, a
second single domain antibody, or a label.
4. (canceled)
5. A nucleic acid molecule encoding the single domain antibody of
claim 1.
6. (canceled)
7. A nucleic acid molecule encoding the fusion protein or conjugate
of claim 3.
8. (canceled)
9. A pharmaceutical composition comprising the antibody of claim 1
and a pharmaceutically acceptable carrier.
10. An isolated cell comprising the nucleic acid molecule of claim
5.
11. A method for inhibiting insulin-like growth factor (IGF)
mediated signaling pathway comprising administering a single domain
antibody comprising: (a) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3; (b) SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; or (c) SEQ ID
NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or a fusion protein or
conjugate comprising the antibody, or a nucleic acid molecule
encoding the antibody.
12. A method for inhibiting the phosphorylation of insulin-like
growth factor receptor (IGF-1R) comprising administering a single
domain antibody comprising: (a) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ
ID NO: 3; (b) SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; or (c)
SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or a fusion protein
or conjugate comprising the antibody, or a nucleic acid molecule
encoding the antibody.
13. A method for treating a cancer associated with the expression
of insulin-like growth factor (IGF) comprising administering a
single domain antibody comprising: (a) SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3; (b) SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7;
or (c) SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or a fusion
protein or conjugate comprising the antibody, or a nucleic acid
molecule encoding the antibody.
14. The method of claim 13, wherein the cancer is selected from the
group consisting of sarcomas, breast cancer, prostate cancer, colon
cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian
cancer, endometrial cancer, melanoma, neuroblastoma, multiple
myeloma, and hepatocellular carcinoma.
15. The method of claim 13, wherein the single domain antibody
comprises SEQ ID NO: 4, 8, or 12.
16. A method of detecting the presence of insulin-like growth
factor (IGF)-2 or IGF-1 receptor (IGF-1R) in a sample comprising
contacting the sample with the single domain antibody of claim 1,
wherein binding of the single domain antibody to IGF-2 or IGF-1R
indicates the presence of IGF-2 or IGF-1R in the sample.
17. A kit for detecting the presence of insulin-like growth factor
(IGF)-2 or IGF-1 receptor (IGF-1R) in a sample comprising the
single domain antibody of claim 1.
18. A method of producing the antibody of claim 1, comprising: (a)
transforming a cell with the nucleic acid molecule encoding the
antibody; (b) culturing the cell in culture medium under conditions
sufficient to express the antibody; and (c) harvesting the antibody
from the cell or culture medium.
19. A vector comprising the nucleic acid molecule of claim 5.
20. A vector comprising the nucleic acid molecule of claim 7.
21. A pharmaceutical composition comprising the fusion protein or
conjugate of claim 3 and a pharmaceutically acceptable carrier.
22. A pharmaceutical composition comprising the nucleic acid
molecule of claim 5 and a pharmaceutically acceptable carrier.
23. The method of claim 11, wherein the single domain antibody
comprises SEQ ID NO: 4, 8, or 12.
Description
BACKGROUND OF THE INVENTION
[0001] Insulin-like growth factor (IGF) 1 receptor (IGF-1R) is a
receptor tyrosine kinase that is widely expressed in human
epithelial cancers (see, e.g., LeRoith et al., Cancer Letters, 195:
127-137 (2003)). The receptor is activated by its cognate ligands,
insulin-like growth factor 1 (IGF-1) and 2 (IGF-2). IGF binding
activates intrinsic tyrosine kinase activity, resulting in receptor
autophosphorylation and stimulation of signaling cascades that
include the IRS-1/PI-3K/AKT/mTOR, and Grb2/Sos/Ras/MAPK pathways.
The IGF mediated signaling has been implicated in the development
of several epithelial cancers, such prostate, breast, and
colorectal cancers. There is a need for cancer therapeutic agents
that target the IGF mediated signaling pathway.
BRIEF SUMMARY OF THE INVENTION
[0002] The invention provides a single domain antibody comprising
(a) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; (b) SEQ ID NO: 5,
SEQ ID NO: 6, and SEQ ID NO: 7; (c) SEQ ID NO: 9, SEQ ID NO: 10,
and SEQ ID NO: 11; (d) SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO:
15; (e) SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; (f) SEQ ID
NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; or (g) SEQ ID NO: 25, SEQ
ID NO: 26, and SEQ ID NO: 27, as well as a nucleic acid encoding
the antibody and cell comprising the nucleic acid.
[0003] The invention also provides a method of inhibiting
insulin-like growth factor (IGF) mediated signaling pathway in a
mammal comprising administering to the mammal a single domain
antibody comprising (a) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3; (b) SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; or (c) SEQ ID
NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, or a fusion protein or
conjugate comprising the antibody, or a nucleic encoding the
antibody, whereby the IGF mediated signaling pathway is
inhibited.
[0004] The invention additionally provides a method of treating a
cancer associated with the expression of IGF in a mammal comprising
administering to the mammal a single domain antibody comprising (a)
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; (b) SEQ ID NO: 5, SEQ
ID NO: 6, and SEQ ID NO: 7; or (c) SEQ ID NO: 9, SEQ ID NO: 10, and
SEQ ID NO: 11, or a fusion protein or conjugate comprising the
antibody, or a nucleic encoding the antibody, whereby the cancer is
treated.
[0005] The invention further provides a method of detecting the
presence of IGF-2 or IGF-1R in a sample comprising contacting the
sample with a single domain antibody comprising (a) SEQ ID NO: 13,
SEQ ID NO: 14, and SEQ ID NO: 15; (b) SEQ ID NO: 17, SEQ ID NO: 18,
and SEQ ID NO: 19; (c) SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO:
23; or (d) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, wherein
binding of the single domain antibody to IGF-2 or IGF-1R indicates
the presence of IGF-2 or IGF-1R in the sample.
[0006] Also provided herein are related compositions, kits, and
methods, such as methods for preparing dAbs.
DETAILED DESCRIPTION OF THE INVENTION
[0007] Provided herein is a single domain antibody (dAb), which is
also known as an engineered antibody domain (eAd), that binds to
human insulin growth factor 1 receptor (IGF-1R) or IGF ligand 2
(IGF-2). Single domain antibodies comprise only a single variable
domain, such as the heavy-chain variable region (V.sub.H) or
light-chain variable region (V.sub.L) (Ward et al., Nature, 341:
544-546 (1989); Holt et al., TRENDS in Biotechnology, 21(11):
484-490 (2003)). The variable region, in turn, comprises
complimentary determining regions (CDRs) that confer binding
specificity, and framework regions, which those parts of the
variable domain other than the CDRs. dAbs are highly expressed in
microbial cell culture, show favorable biophysical properties
including solubility and temperature stability, and are well suited
to selection and affinity maturation by in vitro selection systems
such as phage display. dAbs also are bioactive as monomers and,
owing to their small size and inherent stability, can be formatted
into larger molecules to create drugs with prolonged serum
half-lives or other pharmacological activities.
[0008] The dAb can have any suitable framework region. However, in
a preferred embodiment, the single domain antibody is human or
humanized to lessen the chance that an antibody administered to a
human will evoke an undesirable immune response. Thus, the
framework region of the dAb, desirably, is that of a human variable
domain. Most preferably, the antibody comprises the framework
regions derived from a human heavy-chain variable domain. However,
in some instances, one or more framework (FR) residues of the human
antibody can be replaced by corresponding non-human residues, such
as FR residues from analogous sites in rodent antibodies (Jones et
al., Nature, 321:522-525 (1986), Reichmann et al., Nature,
332:323-327 (1988); Presta, Curr. Opin. Struct. Biol., 2:593-596
(1992)).
[0009] The antibody provided herein comprises three CDRs. In a
first embodiment, the antibody comprises as the CDRs SEQ ID NO: 1,
SEQ ID NO: 2, and SEQ ID NO: 3. One example of such a single domain
antibody, wherein the framework region is that of a human heavy
chain variable domain, is a single domain antibody comprising SEQ
ID NO: 4 (m632).
[0010] In a second embodiment, the single domain antibody comprises
as the CDRs SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. One
example of such a single domain antibody, wherein the framework
region is that of a human heavy chain variable domain, is a single
domain antibody comprising SEQ ID NO: 8 (m636).
[0011] In a third embodiment, the single domain antibody comprises
as the CDRs SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. One
example of such a single domain antibody, wherein the framework
region is that of a human heavy chain variable domain, is a single
domain antibody comprising SEQ ID NO: 12 (m546).
[0012] In a fourth embodiment, the single domain antibody comprises
as the CDRs SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. One
example of such a single domain antibody, wherein the framework
region is that of a human heavy chain variable domain, is a single
domain antibody comprising SEQ ID NO: 16 (m534).
[0013] In a fifth embodiment, the single domain antibody comprises
as the CDRs SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. One
example of such a single domain antibody, wherein the framework
region is that of a human heavy chain variable domain, is a single
domain antibody comprising SEQ ID NO: 20 (m535).
[0014] In a sixth embodiment, the single domain antibody comprises
as the CDRs SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23. One
example of such a single domain antibody, wherein the framework
region is that of a human heavy chain variable domain, is a single
domain antibody comprising SEQ ID NO: 24 (m536).
[0015] In a seventh embodiment, the single domain antibody
comprises as the CDRs SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO:
27. One example of such a single domain antibody, wherein the
framework region is that of a human heavy chain variable domain, is
a single domain antibody comprising SEQ ID NO: 28 (m537).
[0016] The dAb can be fused to another polypeptide or other moiety
to provide fusion protein or conjugate comprising the dAb. For
example, the dAb can be polymerized (fused) to one or more
additional antibodies, antibody fragments (e.g., Fab, single chain,
ScFv, etc.), or dAbs to provide a multivalent antibody construct.
The one or more additional antibodies can be the same or different,
i.e., target the same or different antigen. For instance, two or
more dAbs that bind to a member of the IGF pathway can be fused
together, or a dAb to a member of the IGF pathway can be fused to a
dAb to a different antigen that may be another therapeutic target
or a molecule that performs some other function, such as cell
targeting or enhanced stability (e.g., a dAb to serum albumin).
Alternatively, or in addition, the dAb can be fused to another
immunoglobulin component, such as an Fc domain of a human or
non-human antibody (e.g., human IgG1 Fc), or the dAb can be
conjugated or fused with a toxin, a cell-targeting moiety, a
stabilizing moiety such as polyethylene glycol (PEG) or a molecule
(e.g., peptide) that binds serum albumin, or a label or other
detectable moiety (e.g., a radiolabel, a fluorophore, a
chromophore, an imaging agent, a metal ion, etc.). Such fusion
proteins or conjugates can be produced using standard molecular
biology techniques.
[0017] Preferably, the fusion proteins have molecular weights of
more than 60 kDa (the human kidney filtration limit) but less than
that of full-length antibodies (e.g., 150 kDa for an IgG1), thereby
having a long half-life in circulation while still retaining better
penetration into solid tumors that full-length antibodies.
Accordingly, the fusion proteins of the invention can have a
molecular weight of at least 60 kDa but less than 150 kDa (e.g., 65
kDa, 70 kDa, 75 kDa, 80 kDa, 90 kDa, 95 kDa, 100 kDa, 105 kDa, 110
kDa, 115 kDa, 120 kDa, 125 kDa, 130 kDa, at 135 kDa, 140 kDa, or
145 kDa).
[0018] Any single domain antibody of the invention, whether
attached to other sequences or not, can also include insertions,
deletions, substitutions, or other selected modifications of
particular regions or specific amino acids residues, provided the
activity of the single domain antibody is not significantly altered
or impaired compared to the non-modified single domain antibody.
These modifications can provide for some additional property, such
as to remove/add amino acids capable of disulfide bonding, to
increase its bio-longevity, to alter its secretory characteristics,
etc. In any case, the single domain antibody must possess a
bioactive property, such as specific binding to its cognate
antigen. Functional or active regions of the single domain antibody
may be identified and/or improved by mutagenesis of a specific
region of the protein, followed by expression and testing of the
expressed polypeptide. For example, amino acid sequence variants of
antibodies or antibody fragments can be generated and those that
display equivalent or improved affinity for antigen can be
identified using standard techniques and/or those described herein.
Methods for generating amino acid sequence variants are readily
apparent to a skilled practitioner in the art and can include
site-specific mutagenesis or random mutagenesis (e.g., by PCR) of
the nucleic acid encoding the single domain antibody (Zoller, M. J.
Curr. Opin. Biotechnol. 3: 348-354 (1992)). Both naturally
occurring and non-naturally occurring amino acids (e.g.,
artificially-derivatized amino acids) may be used to generate amino
acid sequence variants of the antibodies and antibody fragments of
the invention.
[0019] Also provided herein is a nucleic acid that encodes a dAb or
fusion polypeptide described herein. Nucleic acids include single
stranded and double stranded nucleic acids of any type (e.g., DNA,
RNA, DNA/RNA hybrid). Such nucleic acids may find use both
therapeutically and in methods of producing the dAb.
[0020] The nucleic acid encoding the antibody or fusion polypeptide
can be part of a vector. Vectors include nucleic acid vectors, such
as naked DNA and plasmids, and viral vectors, such as retroviral
vectors, parvovirus-based vectors (e.g., adenoviral-based vectors
and adeno-associated virus (AAV)-based vectors), lentiviral vectors
(e.g., Herpes simplex (HSV)-based vectors), poxviral vectors (e.g.,
vaccinia virus-based vectors and fowlpox virus-based vectors), and
hybrid or chimeric viral vectors, such as an adenoviral backbone
with lentiviral components (see, e.g., Zheng et al., Nat. Biotech.,
18(2): 176-80 (2000); International Patent Application WO 98/22143;
International Patent Application WO 98/46778; and International
Patent Application WO 00/17376) and an adenoviral backbone with AAV
components (see, e.g., Fisher et al., Hum. Gene Ther., 7: 2079-2087
(1996)). Vectors and vector construction are known in the art (see,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
edition, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et
al., Current Protocols in Molecular Biology, Green Publishing
Associates and John Wiley & Sons, New York, N.Y. (1994)).
[0021] The vector can comprise any suitable promoter and other
regulatory sequences (e.g., transcription and translation
initiation and termination codons, which are specific to the type
of host) to control the expression of the nucleic acid sequence
encoding the polypeptide. The promoter can be a native or nonnative
promoter operably linked to the nucleic acid molecule described
above. The selection of promoters, including various constitutive
and regulatable promoters, is within the skill of an ordinary
artisan. Examples of regulatable promoters include inducible,
repressible, and tissue-specific promoters. Specific examples
include viral promoters, such as adenoviral, vaccinia virus, and
AAV promoters. Additionally, combining the nucleic acid described
above with a promoter is within the skill in the art.
[0022] A cell (e.g., an isolated host cell) comprising the dAb or
nucleic acid molecule encoding the dAb, optionally in the form of a
vector, also is provided, which may be useful, for example, as a
therapeutic agent or for producing a dAb. Any suitable cell can be
used, including prokaryotic and eukaryotic cells. Examples include
host cells, such as E. coli (e.g., E. coli Tb-1, TG-1, DH5.alpha.,
XL-Blue MRF' (Stratagene), SA2821, and Y1090), Bacillus subtilis,
Salmonella typhimurium, Serratia marcescens, Pseudomonas (e.g., P.
aerugenosa), N. grassa, insect cells (e.g., Sf9, Ea4), yeast (S.
cerevisiae) cells, and cells derived from a mammal, including
murine and human cell lines. Specific examples of suitable
eukaryotic host cells include VERO, HeLa, 3T3, Chinese hamster
ovary (CHO) cells, W138 BHK, COS-7, and MDCK cells. Alternatively,
cells from a mammal, such as a human, to be treated in accordance
with the methods described herein can be used as host cells.
Methods of introducing nucleic acids and vectors into isolated host
cells and the culture and selection of transformed host cells in
vitro are known in the art and include the use of calcium
chloride-mediated transformation, transduction, conjugation,
triparental mating, DEAE, dextran-mediated transfection, infection,
membrane fusion with liposomes, high velocity bombardment with
DNA-coated microprojectiles, direct microinjection into single
cells, and electroporation (see, e.g., Sambrook et al., Molecular
Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY
(1989); Davis et al., Basic Methods in Molecular Biology (1986);
and Neumann et al., EMBO J. 1: 841 (1982)). Desirably, the cell
comprising the vector or nucleic acid molecule expresses the
nucleic acid sequence, such that the nucleic acid sequence is
transcribed and translated efficiently by the cell.
[0023] The dAbs described herein, as well as any conjugates or
fusion proteins, can be produced by a method comprising (a)
transforming a cell with a nucleic acid encoding the antibody; (b)
culturing the cell in culture medium under conditions sufficient to
express the antibody; and (c) harvesting the antibody from the cell
or culture medium. The inventive dAbs can be expressed in
prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g.,
yeast, insect, or mammalian cells), and subsequently harvested and
purified, as necessary, using well known methods (see, e.g.,
Sambrook et al. Molecular Cloning: a Laboratory Manual, Cold Spring
Harbor Laboratory Press (1989); and Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y. (2001), which is updated quarterly). Specific techniques for
transforming, culturing, and harvesting the dAb are known in the
art. For example, the dAb can be expressed in a bacterial system,
such as E. coli, or fungal systems, such as yeast (see, e.g., Holt
et al., supra). Alternatively, the dAb can be produced in
mammalian, avian, or plant systems (see, e.g., Holt et al., supra).
If the dAb has poor solubility, the dAb can be expressed in the
form of insoluble inclusion bodies and refolded in vitro (see,
e.g., Holt et al., supra). Other aspects of the method of preparing
a dAb are as described with respect to the dAb and nucleic acid of
the invention.
[0024] Other techniques for antibody production know in the art
also can be used to produce the dAb. Examples of techniques for
antibody production include those described by Cole et al.
(Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985)) and by Boerner et al. (J. Immunol., 147(1): 86-95 (1991)).
Also, dAbs can be produced using phage display libraries
(Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al.,
J. Mol. Biol., 222: 581 (1991); and C. F. Barbas, D. R. Burton, J.
K. Scott, G. J. Silverman, Phage Display: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(2001)).
[0025] The dAb can be used for any purpose. For example, a dAb
comprising (a) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; (b)
SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; or (c) SEQ ID NO: 9,
SEQ ID NO: 10, and SEQ ID NO: 11 can be used to inhibit the IGF
signaling pathway in a cell or in a mammal. Thus, provided herein
is a method of inhibiting the IGF signaling pathway in a cell,
which may be in a mammal, comprising administering to the cell or
mammal the dAb, whereby the IGF signaling pathway is inhibited. The
IGF signaling pathway can be inhibited by any mechanism, without
limitation. Thus, for example, the method of inhibiting the IGF
signaling pathway can comprise inhibiting phosphorylation of the
IGF-1 receptor (IGF-1R).
[0026] Furthermore, the method of inhibiting the IGF signaling
pathway can be used to achieve any end result, such as for the
treatment of a disease associated with IGF overexpression. For
example, the method of inhibiting the IGF signaling pathway can be
performed by administering the dAb, as described above, to a cell
in a mammal afflicted with a disease associated with IGF
overexpression, such as cancer. Through practice of the method, one
or more symptoms of the disease is alleviated and the disease is,
thereby, treated. Treatment of cancer, in particular, can comprise
alleviation of any one or more symptoms of the cancer, including,
without limitation, an inhibition of the growth of cancer cells, a
decrease in metastasis, an increase in cancer cell death, and an
increase in the survival of the mammal afflicted with the
cancer.
[0027] Non-limiting examples of specific types of cancers include
cancer of the head and neck, eye, skin, mouth, throat, esophagus,
chest, bone, lung, colon, sigmoid, rectum, stomach, prostate,
breast, ovaries, kidney, liver, pancreas, brain, intestine, heart
or adrenals. More particularly, cancers include solid tumor,
sarcoma, carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, a blood-born tumor, acute lymphoblastic leukemia,
acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell
leukemia, acute myeloblastic leukemia, acute promyelocytic
leukemia, acute monoblastic leukemia, acute erythroleukemic
leukemia, acute megakaryoblastic leukemia, acute myelomonocytic
leukemia, acutenonlymphocyctic leukemia, acute undifferentiated
leukemia, chronic myelocytic leukemia, chronic lymphocytic
leukemia, hairy cell leukemia, or multiple myeloma. See, e.g.,
Harrison's Principles of Internal Medicine, Eugene Braunwald et
al., eds., pp. 491 762 (15th ed. 2001). The methods of the
invention are believed to be useful for the treatment of sarcomas
(e.g., osteosarcoma and rhabdomyosarcoma), breast, prostate, colon,
lung, pancreatic, cervical, ovarian, and endometrial cancers,
melanoma, neuroblastoma, multiple myeloma, and hepatocellular
carcinoma, as well as any other cancer known to be responsive to
inhibitors of the IGF signaling pathway.
[0028] IGF-1R activity has been linked to other diseases, including
benign prostatic hyperplasia (BPH), diarrhea associated with
metastatic carcinoid and vasoactive intestinal peptide secreting
tumors (e.g., VIPoma or Werner-Morrison syndrome), acromegaly,
gigantism, psoriasis, atherosclerosis, spinocerebellar ataxia and
smooth muscle restenosis of blood vessels or inappropriate
microvascular proliferation, such as that found as a complication
of diabetes, especially of the eye. Thus, the methods of the
invention are believed to be useful for the treatment of such
diseases, as well.
[0029] The single domain antibody can be used alone or in
combination with other anti-cancer therapies, such as chemotherapy
and radiotherapy.
[0030] The single domain antibody can be administered to the mammal
directly, or by administering to the mammal a nucleic acid molecule
encoding the antibody, optionally in a vector, or a cell comprising
the nucleic acid. Nucleic acids, vectors, and cells comprising the
nucleic acids are as previously described herein. Furthermore, the
dAb can be administered alone or as part of a conjugate or fusion
molecule, as previously described.
[0031] The cell can be any type of cell, as discussed elsewhere
herein, especially a cancer cell. When the cell is in a mammal, the
mammal can be any suitable mammal, such as a mouse, rat, guinea
pig, hamster, rabbit, cat, dog, sheep, cow, horse, pig, or primate.
Preferably, the mammal is a human.
[0032] The dAb, conjugate, fusion protein, nucleic acid molecule,
vector, or cells, can be administered to a mammal alone, or in
combination with a carrier (i.e., a pharmaceutically acceptable
carrier). By pharmaceutically acceptable is meant a material that
is not biologically or otherwise undesirable (i.e., the material
can be administered to a mammal, along with the single domain
antibody, nucleic acid, vector, or cell, without causing any
undesirable biological effects or interacting in a deleterious
manner with any of the other components of the pharmaceutical
composition in which it is contained). The carrier is selected to
minimize any degradation of the agent and to minimize any adverse
side effects in the mammal, as would be well-known to one of
ordinary skill in the art.
[0033] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.
R. Gennaro, Mack Publishing Company, Easton, Pa. (1995).
Pharmaceutical carriers, include sterile water, saline, Ringer's
solution, dextrose solution, and buffered solutions at
physiological pH. Typically, an appropriate amount of a
pharmaceutically acceptable salt is used in the formulation to
render the formulation isotonic. The pH of the solution is
preferably from about 5 to about 8 (e.g., about 5.5, about 6, about
6.5, about 7, about 7.5, and ranges thereof). More preferably, the
pH is about 7 to about 7.5. Further carriers include
sustained-release preparations, such as semipermeable matrices of
solid hydrophobic polymers containing the polypeptide, which
matrices are in the form of shaped articles (e.g., films,
liposomes, or microparticles). It will be apparent to those persons
skilled in the art that certain carriers may be more preferable
depending upon, for instance, the route of administration and
concentration of composition being administered.
[0034] Compositions (e.g., pharmaceutical compositions) comprising
the single domain antibody, nucleic acid molecule, vector, or cell
can include carriers, thickeners, diluents, buffers, preservatives,
surface agents, and the like. The compositions also can include one
or more active agents, such as an antimicrobial agent, an
anti-inflammatory agent, an anesthetic, an anti-viral agent, or a
cytotoxic agent (e.g., a chemotherapeutic agent, a small drug, a
prodrug, a taxoid, or a toxin).
[0035] The composition (e.g., pharmaceutical composition)
comprising the single domain antibody, nucleic acid molecule,
vector, or cell can be administered (e.g., to the mammal, a cell, a
tissue, or a tumor) in any suitable manner depending on whether
local or systemic treatment is desired, and on the area to be
treated. Administration can be topically (including ophthalmically,
vaginally, rectally, intranasally, transdermally, and the like),
orally, by inhalation, or parenterally (including by intravenous
drip or subcutaneous, intracavity, intraperitoneal, intradermal, or
intramuscular injection). Topical intranasal administration refers
to the delivery of the compositions into the nose and nasal
passages through one or both of the nares and can comprise delivery
by a spraying mechanism or droplet mechanism, or through
aerosolization of the nucleic acid or vector. Administration of the
compositions by inhalant can be through the nose or mouth via
delivery by a spraying or droplet mechanism. Delivery can also be
directly to any area of the respiratory system (e.g., lungs) via
intubation. Alternatively, administration can be intratumoral.
Local or intravenous injection is preferred.
[0036] If the composition is to be administered parenterally, the
administration is generally by injection. Injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution of suspension in
liquid prior to injection, or as emulsions. Additionally, parental
administration can involve the preparation of a slow-release or
sustained-release system, such that a constant dosage is maintained
(see, e.g., U.S. Pat. No. 3,610,795). Preparations for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils, such as
olive oil, and injectable organic esters, such as ethyl oleate.
Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives also can
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and inert gases and the like.
[0037] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids, and powders. Conventional pharmaceutical carriers;
aqueous, powder, or oily bases; thickeners; and the like may be
necessary or desirable.
[0038] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids, or binders may be desirable.
[0039] Some of the compositions can potentially be administered as
a pharmaceutically acceptable acid- or base-addition salt, formed
by reaction with inorganic acids, such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base, such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases, such as mono-, di-, trialkyl, and
aryl amines and substituted ethanolamines.
[0040] Additionally, probiotic therapies are envisioned by the
present invention. Viable host cells containing the nucleic acid
molecule or vector of the invention and expressing the single
domain antibody can be used directly as the delivery vehicle to the
desired site(s) in vivo. Preferred host cells for the delivery of
the single domain antibody directly to desired site(s), such as,
for example, to a selected body cavity, can comprise bacteria. More
specifically, such host cells can comprise suitably engineered
strain(s) of lactobacilli, enterococci, or other common bacteria,
such as E. coli, normal strains of which are known to commonly
populate body cavities. More specifically yet, such host cells can
comprise one or more selected nonpathogenic strains of
lactobacilli, such as those described by Andreu et al. (J. Infect.
Dis., 171(5): 1237-43 (1995)), especially those having high
adherence properties to epithelial cells (e.g., vaginal epithelial
cells) and suitably transformed using the nucleic acid molecule or
vector of the invention.
[0041] The single domain antibody, conjugate, fusion protein,
nucleic acid molecule, vector, or cell can be administered with a
pharmaceutically acceptable carrier and can be delivered to the
mammal in vivo and/or ex vivo by a variety of mechanisms well-known
in the art. If ex vivo methods are employed, cells or tissues can
be removed and maintained outside the body according to standard
protocols known in the art. The compositions can be introduced into
the cells or tissue via any gene transfer mechanism, such as
calcium phosphate mediated gene delivery, electroporation,
microinjection, or proteoliposomes. The transduced cells then can
be infused (e.g., with a pharmaceutically acceptable carrier) or
homotopically transplanted back into the mammal per standard
methods for the cell or tissue type. Standard methods are known for
transplantation or infusion of various cells into a mammal.
[0042] The exact amount of the single domain antibody, conjugate,
fusion protein, nucleic acid molecule, vector, cell, or
compositions thereof required to elicit the desired effect will
vary from mammal to mammal, depending on the species, age, gender,
weight, and general condition of the mammal, the particular single
domain antibody, nucleic acid molecule, vector, or cell used, the
route of administration, and whether other drugs are included in
the regimen. Thus, it is not possible to specify an exact amount
for every composition. However, an appropriate amount can be
determined by one of ordinary skill in the art using only routine
experimentation given the teachings herein. The dosage ranges for
the administration of the compositions are those large enough to
produce the desired effect; however, the dosage should not be so
large as to cause adverse side effects, such as unwanted
cross-reactions, anaphylactic reactions, and the like. Dosage can
vary, and can be administered in one or more (e.g., two or more,
three or more, four or more, or five or more) doses daily, for one
or more days. Guidance in selecting appropriate doses for
antibodies is found in the literature on therapeutic uses of
antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et
al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and
pp. 303-357; Smith et al., Antibodies in Human Diagnosis and
Therapy, Haber et al., eds., Raven Press, New York (1977) pp.
365-389. A typical daily dosage of the single domain antibody used
alone might range from about 1 .mu.g/kg to up to 100 mg/kg of body
weight or more per day, depending on the factors mentioned
above.
[0043] The invention also includes kits comprising the single
domain antibody, nucleic acid molecule, vector, cell, or
compositions thereof. The kit can include a separate container
containing a suitable carrier, diluent, or excipient. The kit also
can include an adjuvant, cytokine, active agent, immunoassay
reagents, PCR reagents, radiolabels, and the like. Additionally,
the kit can include instructions for mixing or combining
ingredients and/or administration.
[0044] The single domain antibody can be used to detect the
presence of IGF-2 or IGF-1R in a sample. Such a method can comprise
contacting a sample with the single domain antibody, wherein
binding of the single domain antibody to IGF-2 or IGF-1R indicates
the presence of IGF-2 or IGF-1R in the sample. Any suitable sample
can be used, such as blood, serum, cells, or tissue isolated from a
mammal (e.g., a human). Furthermore, the sample can be contacted
with the single domain antibody by any suitable method. For
instance, the antibody can simply be combined with the sample, or
the antibody can be immobilized on a substrate and the sample
applied to the substrate. Thereafter, the substrate can be washed
and examined for the presence of bound antigen. Other common
techniques also can be used. Similarly, the binding of the antibody
to the antigen can be detected by routine techniques, including the
use of labels and/or probes to detect bound antigen or
antigen-antibody complexes. All other aspects of the method are as
previously described with respect to the single domain antibody of
the invention.
[0045] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
[0046] This example demonstrates the design and construction of a
large phage-displayed domain antibody (dAb) library.
[0047] The m81 library previously was constructed based on highly
stable, soluble VH scaffold, m0, by grafting in vivo formed CDR2
(H2) and CDR3 (H3), and randomizing four putative solvent
accessible residues in the CDR1 (H1) of m0 to A, D, S, and Y, which
are residues most widely used in human antibodies (see, e.g., Chen
et al., Methods Mol. Biol., 525: 81-99 (2009); and Chen et al., J.
Mol. Biol., 382: 779-789 (2008)). In order to increase diversity
and minimize immunogenicity, a human antibody light chain CDR3 (L3)
repertoire was grafted into the H1 of the m81 library, which
resulted in the generation of a new library (designated m91), which
combined the most diversified CDRs of an antibody (L3, H2, and H3)
in the same scaffold.
[0048] To efficiently amplify highly diverse L3 repertoires,
primers based on human germline VL sequences were designed. The
sense primers targeted the last three residues of the light chain
FR3 (LFR3) that are highly conserved among different families of
kappa (KL) and lambda (LL) light chains. The antisense primers
targeted the first three residues of the J gene product. Thus, the
PCR products contained L3 plus an additional three residues from
LFR3.
[0049] Since the amplification of L3 was inefficient when cDNA or
plasmid DNA was used directly as a template, full-length KL and LL
first were amplified under standard conditions (using primers
described in Zhu et al., Methods Mol. Biol., 525: 129-142 (2009)),
gel purified, and then used as templates for L3 amplification.
Because the last residue of LFR3 (position 104, IMGT annotation) is
cysteine in almost all germline sequences (which could affect
protein folding leading to a low yield of properly folded
antibodies), the cysteine was mutated to serine or glycine
following the amplification and pooling of the kappa (KL3) and
lambda (LL3) L3 repertoires. The two other residues of LFR3
(positions 102 and 103, IMGT annotation) are most frequently are
tyrosine, which is highly hydrophobic. To reduce the possibility of
antibody aggregation, the tyrosine at position 102 (IMGT
annotation) was mutated to aspartic acid, asparagine, or histidine.
Low annealing temperature (40.degree. C.) was used from the PCR
mutagenesis to allow for efficient secondary amplification of the
L3 fragments containing the desired mutation.
[0050] For assembly of full-length VHs, human H2 and H3 repertoires
and the FR2, FR3, and FR4 of m0 were amplified as a whole from the
m81 library. The FR1 of m0 also was PCR-amplified from the m81
plasmid DNA and joined to the L2 repertoire by overlapping PCR. The
entire chimeric VHs were assembled by further joining the L3
repertoire to the H2 and H3 repertoires. The products were cloned
into phagemid pComb3X and a large (about 10.sup.10) library
(designated m91) was obtained by performing 100
electroporations.
[0051] To estimate the degree of diversity of the m91 library, 126
randomly selected dAb clones were analyzed for their gene usage,
somatic mutations, CDR length, and how different CDRs are combined.
There were no identical L3 and H3 sequences found. H2s were less
diverse with three groups (with 19, 4, and 3 members, respectively)
of sequences containing identical H2s from VH families 7, 4, and 4,
respectively. Of the 126 L3 sequences, 88 were derived from all
families of KL except family 7. About 75% of the 88 sequences were
from family 1 of KL. The sequences varied in length from 10 to 14
residues and more than 90% were 10 residues in length.
[0052] The remaining 38 L3 sequences were from only 2 of 11 LL
families. More than 80% of the 38 sequences were from family 1. The
L3 sequences varied in length from 9 to 12 residues. Most of the L3
sequences (82.5%) were mutated in their V genes compared to the
closest corresponding germline genes. 57% contained two or more
mutations in their amino acid sequences.
[0053] The distribution and somatic mutations of H2s in m91 were
consistent with those in m81 except that in m91, H2 gene usage was
biased slightly toward VH1 and VH7, while VH2- and VH6-derived
sequences were absent.
[0054] The H3 lengths of dAbs from m91 ranged from 5 to 23
residues. The distribution was in agreement with the reported
frequency in m81, but there was an increased number of H3s with
lengths shorter than 9 residues and longer than 18 residues.
[0055] In order to determine the combinatorial diversity of the
library, the pairing between the L3 origin, H2 origin, and H3
length was plotted. Regardless of the preferential amplification of
gene fragments from certain families, the CDRs were paired
randomly. These results indicate a high degree of diversity in the
m91 library.
[0056] In order to analyze the dAbs from the m91 library for
certain biophysical properties such as oligomerization,
aggregation, and degradation, four dAbs were randomly selected and
more extensively characterized. After purification on
Ni-nitrilotriacetic acid resin, the four dAbs were dialyzed against
phosphate buffered saline (PBS, pH 7.4, concentrated, and subjected
to long-term storage at 4.degree. C. No precipitation was observed
with these four dAb solutions immediately after purification and
concentration. After storage for about one year, one dAb showed
obvious precipitation (pellet) after centrifugation. Supernatent
fractions were collected and measure for optical density at 280 nm.
No significant degradation was observed. When run on reducing
SDS-PAGE, the four dAbs had apparent molecular weights of about 16
kDa, which is similar to the calculated molecular weights of 15-17
kDa. Two of the dAbs ran faster than the others on a native PAGE
with an apparent molecular weight that is much lower than their
calculated molecule weight, suggesting that these dAbs may fold
more tightly.
[0057] The oligomerization of these dAbs was measured by
size-exclusion chromatography on a Superdex-75 column. The dAbs
were eluted as mono-disperse symmetric peaks, indicating that the
dAbs did not stick to the column matrix. Only one of the dAbs
eluted at the expected size of a monomer, which variations in the
apparent molecular weight were observed with the other dAbs, which
eluted more rapidly or slowly Interestingly, the elution of two of
these dAbs, as well as m36, which is a well-characterized monomeric
dAb (with a calculated molecular weight of about 15 kDa) from the
m81 library, was further delayed in the presence of 300 nM NaCl,
suggesting an increased hydrophobicity of these antibodies. These
results suggest that randomly selected dAbs from the m91 library
generally are stable against aggregation, but may exhibit
variations in their apparent molecular weights.
[0058] It was observed that staphylococcal protein A (SPA) binds to
the VH domain containing the VH3 gene products. Therefore, the
library was panned against SPA in order to investigate possible
conformational changes in the scaffold caused by the grafted L3s.
Forty-six and 43 clones were picked randomly from the third and
fourth round of panning, respectively, sequenced, and analyzed for
L3 and H2 gene usage and 113 length. There was an increased number
(70%, 89%, and 91% for the original library, the third round, or
the fourth round, respectively) of antibodies with KL3s. The
frequencies of V and J gene usages in the KL3s selected after
panning were comparable with those for the original library. The
frequency of antibodies composed of VH3-derived H2 was increased
dramatically (4- to 5-fold) and their H3s were diverse with lengths
ranging from 7 to 20 residues. These results suggest that the
VH3-based scaffold used in the library, m0, preserves its
conformational integrity after grafting of KL3s from almost all
families, as evaluated by SPA binding activity.
EXAMPLE 2
[0059] This example describes the isolation of several fully
humanized dAbs against human IGF-1R and IGF ligand 2 (IGF-2).
[0060] The libraries described in Example 1 (m81 and m91) were
screened for high affinity binding to IGF-1R and/or IGF-2. Several
fully humanized domain antibodies were isolated. The domain
antibodies were one magnitude smaller than regular IgG and were
fully human. As a result, the antibodies have better solid tumor
penetration capability compared with regular IgG and minimal toxic
effects and immunogenicity.
[0061] Two antibodies designated m632 (corresponding to SEQ ID NO:
4) and m636 (corresponding to SEQ ID NOs: 8) were selected from
library m91. m632 and m636 bound with high affinity to IGF-2 as
determined by ELISA. m636 was cross-reactive for both IGF-1 and
IGF-2.
[0062] Five antibodies designated m546 (corresponding to SEQ ID NO:
12), m534 (corresponding to SEQ ID NO: 16), m535 (corresponding to
SEQ ID NO: 20), m536 (corresponding to SEQ ID NO: 24), and m537
(corresponding to SEQ ID NO: 28) bound with high affinity to IGF-1R
as determined by ELISA. m534 and m535 were selected from library
m81. m536, m537, and m546 were selected from library m91.
[0063] The sequences of identified antibodies, as well as the
respective CDRs, are set forth in Table 1.
TABLE-US-00001 TABLE 1 Sequences of dAbs. SEQ ID Ab SEQUENCE NO
m632 QVQLVQSGGGLVQPGGSLRLSCAASDYSQQYKTYPLTFMSW 4
VRQAPGQRLEWVAGISGSGGTTVYADSVKGRFTISRDNSKNT
LYLQMNTLRAEDTAMYYCARVASRDYFDYWGQGTLVTVSS CDR1 QQYKTYPLTF 1 CDR2
ISGSGGTT 2 CDR3 ARVASRDYFDY 3 m636
QVQLVQSGGGLVQPGGSLRLSCAASYYSLQHDNFPYTFMSW 8
VRQAPGQRLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNT
LYLQMNTLRAEDTAMYYCARIRWLQDLDYWGQGTLVTVSS CDR1 LQHDNFPYTF 5 CDR2
ISGSGGST 6 CDR3 ARIRWLQDLDY 7 m546
QVQLVQSGGGLVQPGGSLRLSCAASYYSQQYNSYPITFMSW 12 VRQAPGQRLEWVAS
INQDGSQIDYAGSVKGRFTISRDNSKNTLYLQMNTLRAEDTA
TYYCAVDLRSGARNFQHWGQGTLVTVSS CDR1 QQYNSYPITF 9 CDR2 INQDGSQI 10
CDR3 AVDLRSGARNFQH 11 m534 QVQLVQSGGGLVQPGGSLRLSCAASDFYFYDYEMSWVRQA
16 PGKGLEWIGSISHGGITHYTYSLKSRVTISRDNSKNTLYLQMN
TLRAEDTAMYYCARDYGYAFDIWGQWTTGTVSS CDR1 DFYFYDYE 13 CDR2 ISHGGIT 14
CDR3 ARDYGYAFDI 15 m535 QVQLVQSGGGLVQPGGSLRLSCAASSFSFSDYEMSWVRQAP
20 GKGLEWVAHINSDGVIQYADSVKGRFTISRDNSKNTLYLQM
NTLRAEDTATYYCVRVAVPGKRYFQYWGQGTTVTVSS CDR1 SFSFSDYE 17 CDR2 INSDGVI
18 CDR3 VRVAVPGKRYFQY 19 m536
QVQLVQSGGGLVQPGGSLRLSCAASYYGQSFDSDSVVFMSW 24
VRQAPGKGLEWISSMSNTGTYIDYADSVKGRFTISRDNSKNT
LYLQMNTLRAEDTATYYCVKEWDRGLRRLQHWGQGTVVT VSS CDR1 QSFDSDSVVF 21 CDR2
MSNTGTYI 22 CDR3 VKEWDRGLRRLQH 23 m537
QVQLVQSGGGLVQPGGSLRLSCAASNYSQQTYSAPITFMSW 28
VRQAPGQGLEWVSSTSWNGGTTDYADSVKGRFTISRDNSKN
TLYLQMNTLRAEDTAMYYCVTDTSGWRYFQDWGQGTLVT VSS CDR1 QQTYSAPITF 25 CDR2
TSWNGGTT 26 CDR3 VTDTSGWRYFQD 27
EXAMPLE 3
[0064] This example demonstrates the characterization of the dAbs
identified in Example 2.
[0065] dAbs m632, m636, and m546 were shown to inhibit
phosphorylation of IGF-1R, the first step in the IGF mediated
pathway, which indicates their utility in human IGF related cancer
therapy. In a first experiment, overnight-starved MCF-7 cells
(human breast cancer cell line) were treated with 10 nM IGF-2 and
various concentrations of IGF-2 dAb (m636) or dAb-Fc fusion protein
(m632Fc) ranging from 0.4 to 400 nM. In a second experiment, the
overnight-starved MCF-7 cells were treated with 2 nM IGF-1 and
various concentrations of IGF-1R dAb-Fc fusion protein (m546Fc)
ranging from 2-200 nM.
[0066] The IGF-1R beta subunit was immunoprecipitated, and
tyrosine-phosphorylated IGF-1R (Pi IGF-1R) was detected with
Western blot. The same membrane was re-probed with IGF-1R Ab to
demonstrate the total amount of IGF-1R per lane of the Western
blot. These experiments demonstrated that m636 and m632Fc
significantly inhibited IGF-2-induced phosphorylation of IGF-1R in
MCF-7 cells, while m546Fc significantly inhibited IGF-1-induced
phosphorylation of IGF-1R.
[0067] In contrast, dAbs m534, m535, m536, and m537 did not
significantly inhibit phosphorylation of IGF-1R. In a first
experiment, overnight starved MCF-7 cells were treated with 2 nM
IGF-1 and various concentrations of IGF-1R dAbs (m534, m535, m536,
or m537) ranging from 2 to 200 nM. In a second experiment,
overnight starved MCF-7 cells were treated with 2 nM IGF-1 and
various concentrations of m535Fc fusion protein.
[0068] The IGF-1R beta subunit was immunoprecipitated, and
tyrosine-phosphorylated IGF-1R (Pi IGF-1R) was detected with
Western blot. The same membrane was re-probed with IGF-1R Ab to
demonstrate the total amount of IGF-1R per lane of the Western
blot. These experiments demonstrated that m534, m535, m536, m537,
and m535Fc did not significantly inhibit IGF-1-induced
phosphorylation of IGF-1R on MCF-7 cells.
[0069] Although dAbs m534, m535, m536, and m537 did not inhibit the
IGF signaling pathway, they showed high affinity binding to IGF-1R
(as described in Example 2). Thus, dAbs m534, m535, m536, and m537
can be used as diagnostic or research agents, such as drug
carriers, since the antibodies did not interrupt the IGF signaling
pathway's normal biological functions and, thus, would cause no
side-effects to the human body.
EXAMPLE 4
[0070] This example demonstrates the treatment of cancer using the
inventive antibodies.
[0071] A mouse model of neuroblastoma is prepared using standard
methods. The IGF-IR dAb designated m546 is administered to the
mouse model, and the effect of the dAb upon the growth of the
neuroblastoma cells is observed.
[0072] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0073] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0074] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
28110PRTArtificial SequenceSynthetic 1Gln Gln Tyr Lys Thr Tyr Pro
Leu Thr Phe1 5 1028PRTArtificial SequenceSynthetic 2Ile Ser Gly Ser
Gly Gly Thr Thr1 5311PRTArtificial SequenceSynthetic 3Ala Arg Val
Ala Ser Arg Asp Tyr Phe Asp Tyr1 5 104123PRTArtificial
SequenceSynthetic 4Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Tyr
Ser Gln Gln Tyr Lys 20 25 30Thr Tyr Pro Leu Thr Phe Met Ser Trp Val
Arg Gln Ala Pro Gly Gln 35 40 45Arg Leu Glu Trp Val Ala Gly Ile Ser
Gly Ser Gly Gly Thr Thr Val 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser65 70 75 80Lys Asn Thr Leu Tyr Leu
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr 85 90 95Ala Met Tyr Tyr Cys
Ala Arg Val Ala Ser Arg Asp Tyr Phe Asp Tyr 100 105 110Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120510PRTArtificial
SequenceSynthetic 5Leu Gln His Asp Asn Phe Pro Tyr Thr Phe1 5
1068PRTArtificial SequenceSynthetic 6Ile Ser Gly Ser Gly Gly Ser
Thr1 5711PRTArtificial SequenceSynthetic 7Ala Arg Ile Arg Trp Leu
Gln Asp Leu Asp Tyr1 5 108123PRTArtificial SequenceSynthetic 8Gln
Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Tyr Ser Leu Gln His Asp
20 25 30Asn Phe Pro Tyr Thr Phe Met Ser Trp Val Arg Gln Ala Pro Gly
Gln 35 40 45Arg Leu Glu Trp Val Ser Gly Ile Ser Gly Ser Gly Gly Ser
Thr Tyr 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser65 70 75 80Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu
Arg Ala Glu Asp Thr 85 90 95Ala Met Tyr Tyr Cys Ala Arg Ile Arg Trp
Leu Gln Asp Leu Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120910PRTArtificial SequenceSynthetic 9Gln Gln Tyr
Asn Ser Tyr Pro Ile Thr Phe1 5 10108PRTArtificial SequenceSynthetic
10Ile Asn Gln Asp Gly Ser Gln Ile1 51113PRTArtificial
SequenceSynthetic 11Ala Val Asp Leu Arg Ser Gly Ala Arg Asn Phe Gln
His1 5 1012125PRTArtificial SequenceSynthetic 12Gln Val Gln Leu Val
Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Tyr Tyr Ser Gln Gln Tyr Asn 20 25 30Ser Tyr Pro
Ile Thr Phe Met Ser Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Arg Leu
Glu Trp Val Ala Ser Ile Asn Gln Asp Gly Ser Gln Ile Asp 50 55 60Tyr
Ala Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser65 70 75
80Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr
85 90 95Ala Thr Tyr Tyr Cys Ala Val Asp Leu Arg Ser Gly Ala Arg Asn
Phe 100 105 110Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125138PRTArtificial SequenceSynthetic 13Asp Phe Tyr Phe Tyr
Asp Tyr Glu1 5147PRTArtificial SequenceSynthetic 14Ile Ser His Gly
Gly Ile Thr1 51510PRTArtificial SequenceSynthetic 15Ala Arg Asp Tyr
Gly Tyr Ala Phe Asp Ile1 5 1016116PRTArtificial SequenceSynthetic
16Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Phe Tyr Phe Tyr Asp
Tyr 20 25 30Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Ser Ile Ser His Gly Gly Ile Thr His Tyr Thr Tyr
Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu65 70 75 80Gln Met Asn Thr Leu Arg Ala Glu Asp Thr
Ala Met Tyr Tyr Cys Ala 85 90 95Arg Asp Tyr Gly Tyr Ala Phe Asp Ile
Trp Gly Gln Trp Thr Thr Gly 100 105 110Thr Val Ser Ser
115178PRTArtificial SequenceSynthetic 17Ser Phe Ser Phe Ser Asp Tyr
Glu1 5187PRTArtificial SequenceSynthetic 18Ile Asn Ser Asp Gly Val
Ile1 51913PRTArtificial SequenceSynthetic 19Val Arg Val Ala Val Pro
Gly Lys Arg Tyr Phe Gln Tyr1 5 1020119PRTArtificial
SequenceSynthetic 20Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Ser Phe
Ser Phe Ser Asp Tyr 20 25 30Glu Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala His Ile Asn Ser Asp Gly Val Ile
Gln Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Thr Leu Arg
Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Val 85 90 95Arg Val Ala Val Pro
Gly Lys Arg Tyr Phe Gln Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 1152110PRTArtificial SequenceSynthetic 21Gln Ser
Phe Asp Ser Asp Ser Val Val Phe1 5 10228PRTArtificial
SequenceSynthetic 22Met Ser Asn Thr Gly Thr Tyr Ile1
52313PRTArtificial SequenceSynthetic 23Val Lys Glu Trp Asp Arg Gly
Leu Arg Arg Leu Gln His1 5 1024125PRTArtificial SequenceSynthetic
24Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Tyr Gly Gln Ser Phe
Asp 20 25 30Ser Asp Ser Val Val Phe Met Ser Trp Val Arg Gln Ala Pro
Gly Lys 35 40 45Gly Leu Glu Trp Ile Ser Ser Met Ser Asn Thr Gly Thr
Tyr Ile Asp 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser65 70 75 80Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
Leu Arg Ala Glu Asp Thr 85 90 95Ala Thr Tyr Tyr Cys Val Lys Glu Trp
Asp Arg Gly Leu Arg Arg Leu 100 105 110Gln His Trp Gly Gln Gly Thr
Val Val Thr Val Ser Ser 115 120 1252510PRTArtificial
SequenceSynthetic 25Gln Gln Thr Tyr Ser Ala Pro Ile Thr Phe1 5
10268PRTArtificial SequenceSynthetic 26Thr Ser Trp Asn Gly Gly Thr
Thr1 52712PRTArtificial SequenceSynthetic 27Val Thr Asp Thr Ser Gly
Trp Arg Tyr Phe Gln Asp1 5 1028124PRTArtificial SequenceSynthetic
28Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Asn Tyr Ser Gln Gln Thr
Tyr 20 25 30Ser Ala Pro Ile Thr Phe Met Ser Trp Val Arg Gln Ala Pro
Gly Gln 35 40 45Gly Leu Glu Trp Val Ser Ser Thr Ser Trp Asn Gly Gly
Thr Thr Asp 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser65 70 75 80Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
Leu Arg Ala Glu Asp Thr 85 90 95Ala Met Tyr Tyr Cys Val Thr Asp Thr
Ser Gly Trp Arg Tyr Phe Gln 100 105 110Asp Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120
* * * * *